PROJECT SUMMARY Dengue virus (DENV) is a mosquito-borne flavivirus that infects over 390 million people worldwide annually, primarily in developing nations. There are currently no antiviral treatments for DENV and prevention efforts rely on local control of the mosquito vector, Aedes aegypti. Although efforts to develop a DENV vaccine have been pursued for over 80 years, a safe and effective vaccine remains elusive largely due to the unique pathogenic features of DENV infection. Recent concerns regarding the safety of Dengvaxia (Sanofi Pasteur) has highlighted the need for novel vaccine strategies for DENV. Recent research from several groups has implicated the viral non-structural protein 1 (NS1) in the development of vascular leakage and progression to severe disease. NS1 is produced early in infection and is secreted in large quantities from infected cells into the blood where it goes on to cause plasma leakage by disruption of endothelial cells in the vasculature, mediated through direct interaction with endothelial cells and also indirect action by eliciting immune cells to produce cytokines that cause plasma leakage. Indeed, immunization with recombinant NS1 protein or modified NS1 proteins can protect against NS1-mediated vascular leakage. However, some antibodies produced against NS1 have cross-reactivity to host proteins on endothelial cells and platelets, which are hypothesized to further contribute to pathogenesis in the host. For this reason, NS1 is a promising candidate for a DENV vaccine, but for safety reasons care should be taken to avoid eliciting potentially harmful auto-reactive antibodies. Here, we propose to use highly immunogenic bacteriophage virus-like particle (VLP) platforms to display short NS1 peptides as a novel vaccine strategy. This approach holds promise for eliciting high-titer, long-lasting antibodies against NS1 that are specific for epitopes that do not elicit dangerous cross-reactive antibodies. In Aim 1, we will engineer bacteriophage VLP-based immunogens and immunize mice to elicit antibodies. In Aim 2, we will assess the binding characteristics of antibodies elicited by our bacteriophage VLPs displaying NS1 peptides. In Aim 3, we will perform in vitro assessments of the antibodies elicited by the vaccine candidates, including their ability to block NS1-mediated endothelial cell disruption and immune-cell cytokine production. In Aim 4, we will perform an in vivo assessment of our vaccine candidates using a mouse model of dengue virus disease. Overall these studies will establish the functions of epitope-specific antibodies against NS1 and lead to the identification of vaccines against NS1 for eliciting protective and safe antibody responses.